Chemiluminescence from UVA–exposed skin: Separating photo-induced chemiluminescence from photophysical light emission

https://doi.org/10.1016/j.jphotobiol.2012.05.022Get rights and content

Abstract

Several previous studies have reported luminescence emission from skin following exposure to UVA radiation in air. We show that UVA irradiation of biomaterials and polymers in oxygen, including bovine stratum corneum, followed by photon counting results in a complex emission due to a combination of photophysical processes together with photo-induced chemiluminescence (PICL). The photophysical processes include fluorescence, phosphorescence and charge-recombination luminescence. By irradiating materials in an inert atmosphere such as nitrogen and allowing photophysical light emission to fully decay before admitting oxygen, the weak photo-induced chemiluminescence generated via free radical reactions with oxygen can be separated and analysed. PICL emission from bovine stratum corneum is weaker than for wool keratin and bovine skin collagen, probably due to its higher water content, and the presence of the natural antioxidants ascorbate and tocopherol.

Highlights

► Weak luminescence from UVA-irradiated biomaterials in air is a composite emission. ► The emission includes photophysical processes and chemiluminescence (PICL). ► Photo-induced chemiluminescence can be separated using an experimental protocol. ► PICL emission from bovine stratum corneum is reported for the first time. ► PICL from stratum corneum is weaker than keratin and collagen proteins.

Introduction

Although UVB radiation (280–320 nm) is the major cause of erythema, DNA damage and skin cancer, exposure to UVA radiation (320–400 nm) is also of concern due to its higher intensity in natural sunlight (by one to two orders of magnitude) and its deeper penetration into the skin. A number of studies have reported increased levels of oxidative stress following exposure of skin to UVA radiation using a luminescence technique that is becoming more widely used [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Such studies involve exposure of an area of depilated animal or human skin to UVA, followed by measurement of the intrinsic luminescence emission from the exposed area using photon counting techniques. An important advantage of this approach is that studies can be carried out in vivo.

The first reported use of this technique used the depilated dorsal skin of female Swiss mice [1]. The mice were anaesthetised and shaved skin areas were irradiated with UVA for up to 2 h before moving the mice to a photon counter. A thirteen-fold increase in the intrinsic luminescence intensity of skin was observed after 45–60 min of UVA exposure. Subsequently in vivo experiments were carried out on human forearm skin using a similar approach, with large increases in luminescence observed [4]. On human skin the luminescence intensity was significantly reduced following tape stripping, a widely used method for removing the surface layer of the stratum corneum, suggesting that the stratum corneum is a major source of luminescence from UVA-irradiated skin [4]. The major role of the stratum corneum in generating chemiluminescence was confirmed in a subsequent study, with the intensity decreasing to 10% of the original value following five tape strippings after UVA exposure [11]. More recently the luminescence technique has been used to determine the effectiveness of various antioxidants, topical applications and drugs on oxidative stress in skin [7], [8], [10], [12]. It has been claimed that the origin of the luminescence (or ultraweak photon emission) from irradiated skin occurs via the formation of protein carbonyls and oxidised amino acid residues, in particular Phe, Trp, His and Cys, with weaker emission from Lys and Thr [5].

One of our interests is in the photo-induced chemiluminescence (PICL) of biomaterials and polymers which is emitted by many different organic materials following exposure to UV or visible light. Thermal chemiluminescence (TCL) is already widely used to study the free radical degradation of polymers at elevated temperatures [13], [14], and we have modified a commercial TCL instrument to allow irradiation of samples with UVA or visible light in situ at constant temperature under a controlled atmosphere [15]. We have previously reported on PICL emission from the fibrous proteins wool and feather keratin, silk fibroin and collagen, including cotton and synthetic fibres [15], and the effects of dyes on PICL emission from polymers [16]. In this paper we report on the total luminescence and PICL emission from bovine stratum corneum obtained from bovine hooves [17] following exposure to UVA radiation. The stratum corneum is a water-impermeable barrier layer consisting of corneocytes containing a mixture of α–keratin intermediate filaments (KIF) and matrix composed of intermediate filament associated proteins (IFAPs) [18]. The corneocytes are separated by a lipid-rich intercellular complex. They also contain a network of antioxidants including significant levels of ascorbate and tocopherol. It was of interest to compare the PICL emission from wool with that from stratum corneum, and also to critically compare our data with previous studies on the intrinsic luminescence of UV-exposed skin.

Throughout this paper, ‘total luminescence’ refers to the luminescence emission from materials following UVA-irradiation in oxygen or air. Total luminescence is a complex composite emission of light which occurs by a number of different pathways. These include the photophysical decay processes of fluorescence, phosphorescence and charge recombination luminescence, together with photochemical PICL emission due to reactions of the free radicals formed in the irradiated material with oxygen. A protocol for separation of the light emission due to PICL, the intensity of which is of particular interest since it is related to the level of oxidative stress in biomaterials, from the photophysical emission processes is described.

Section snippets

Materials

Bovine stratum corneum was obtained from the posterior region of bovine hooves as previously described [17]. The Malpighian layer and the outermost exposed surface were dissected exposing the white stratum corneum in a homogeneous layer about 5 mm thick. Thin slices of this material (∼0.5–1 mm thickness) were used to cut samples for photo-induced chemiluminescence studies. Merino wool fabric (100%) was obtained from Armitage Ltd (UK), scoured in warm water containing a few drops of sodium lauryl

Results and discussion

The methodology used by other researchers was followed where the irradiation and photon counting from irradiated skin was carried out in the presence of oxygen [1], [4], [5]. The luminescence emission of bovine stratum corneum after UVA irradiation in O2 for 2 minutes at 35 °C is shown in Fig. 2. For comparison Fig. 2 also shows the luminescence emission from keratin, the major protein component of the stratum corneum, and bovine skin collagen which is the major protein of the dermis under

Conclusions

After exposure to UVA radiation in air or oxygen, bovine stratum corneum produces luminescence which is a composite emission of light from photophysical processes and protein chemiluminescence. It produces weak PICL emission which can be separated from other processes by adopting a protocol where the sample is irradiated in an inert atmosphere and oxygen is admitted after the photophysical luminescence emission has decayed back to zero. Keratin, the major protein component of stratum corneum,

Acknowledgements

We are heavily indebted to Dr Maxine McCall of CSIRO Food and Nutritional Sciences, North Ryde, Sydney for stimulating discussions which led to this research being conducted. We acknowledge the assistance of Dr Jerome Werkmeister at CSIRO Materials Science and Engineering, Clayton, Melbourne for providing the sample of purified bovine skin collagen Type I.

References (43)

  • O.Y. Hao et al.

    A chemiluminescence study of UVA-induced oxidative stress in human skin in vivo

    Journal of Investigative Dermatology

    (2004)
  • F. Khabiri et al.

    Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)-measurement. I: mechanisms of UPE of biological materials

    Skin Research and Technology

    (2008)
  • R. Hagens et al.

    Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin

    Skin Research and Technology

    (2008)
  • A. Jain et al.

    Antioxidant efficacy on human skin in vivo investigated by UVA-induced chemiluminescence decay analysis via induced chemiluminescence of human skin

    Skin Pharmacology and Physics

    (2010)
  • A. Rastogi et al.

    Ultra-weak photon emission as a non-invasive tool for monitoring of oxidative processes in the epidermal cells of human skin: comparative study on the dorsal and the palm side of the hand

    Skin Research and Technology

    (2010)
  • A. Rastogi et al.

    Spontaneous ultraweak photon emission imaging of oxidative metabolic processes in human skin: effect of molecular oxygen and antioxidant defense system

    Journal of Biomedical Optics

    (2011)
  • H. Nishimura et al.

    Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods

    Experimental Dermatology

    (2006)
  • M. Rohr et al.

    Influence of repetitive UVA stimulation on skin protection capacity and antioxidant efficacy

    Skin Pharmacology and Physics

    (2011)
  • I. Blakey et al.

    Chemiluminescence as a probe of polymer oxidation

    Australian Journal of Chemistry

    (2006)
  • A.R. Ugel, W. Idler, Stratum granulosum – dissection from cattle hoof epidermis, Journal of Investigative Dermatology...
  • L.N. Jones

    Protein composition of mammalian stratum corneum

  • Cited by (0)

    View full text